PROJECT SUMMARY There is a fundamental gap in our understanding of why only certain vaccines promote formation of long-lasting and protective antibody responses. Dendritic cells (DC) are professional antigen presenting cells that regulate adaptive immune responses; thus, we think that understanding the role DCs in regulating B cell responses will be key to the development of universal vaccines targeting influenza and HIV. Our experimental data suggest that the major limitations of the relatively ineffective vaccines currently in use or in development might be that: 1) they do not specifically target Langerhans cells (LCs) and 2) the antigen dose is not tailored to maximize the activation of the LCs. Our long-term goal is to understand the role of different DC populations in humoral immune responses and to use that knowledge to design DC-subset-tailored vaccines, with a special focus on influenza and HIV. In pursuit of that goal, the objectives of this application are to: 1) define the mechanisms by which steady state LCs promote germinal center (GC)-dependent humoral immune responses; 2) to characterize the regulatory loop by which high antigen dose and CD103+ DCs inhibit GC-dependent responses induced by LCs; and 3) to test the feasibility of the DC-subset-tailored influenza vaccine. Our central hypothesis is that LCs drive functionally distinct Tfh cells in an antigen dose-dependent manner that ultimately determines whether humoral immune responses are mounted or not. The rationale for the proposed research is that, once the details of how LCs initiate humoral immune responses and regulate each other are known, the process can likely be manipulated to yield innovative approaches for prevention and treatment of a variety of diseases. Guided by strong preliminary data, our hypothesis will be tested in these three Specific Aims: 1. Define the mechanism by which LCs promote GC-dependent humoral immune responses; 2) Determine the mechanism by which high antigen dose and CD103+ DCs inhibit LC-induced GC-dependent humoral immune responses; and 3) Determine the effect of antigen dose on development of protective anti-viral responses. For Aim 1, a well characterized targeting approach will be combined with LC-specific knock out transgenic mouse models, bone marrow chimeras, imaging, in vitro cell cultures and flow cytometry to define the molecular requirements of LC-induced humoral immune responses. In Aim 2, in vitro co-culture experiments and in vivo adoptive cell transfer systems will be used to characterize how high antigen dose and CD103+ DCs regulate LC-induced immune responses. In Aim 3, we will immunize mice through LCs with different doses of flu-HA and then challenge with live influenza to determine the feasibility of the antigen-tailored influenza vaccine. The proposed hypothesis and experimental models are highly novel and innovative. This project is significant because it will advance and expand our understanding on how humoral immune responses are induced and regulated by DC subsets, which we expect will overcome problems that have hindered our ability to generate effective influenza vaccines.